The oculocerebrorenal syndrome of Lowe protein (Ocrl) regulates intracellular traffickin

The levels of phosphatidylinositol second messengers are controlled spatially and temporally by kinases and phosphatases. Inherited mutations in inositol phosphatases can lead to diseases such as myotubular myopathies and Lowe syndrome. The focus of this dissertation is the cellular function of the inositol polyphosphate 5-phosphatase Ocrl which is mutated in patients with Lowe syndrome. The clincial features of this disorder include cataracts and glaucoma, renal Fanconi syndrome, and mild mental retardation. First, the possibility that abnormal trafficking of lysosomal enzymes in patients is responsible for the complex phenotype of Lowe syndrome was tested. Patients were found to have 1.6-2 fold higher levels of plasma lysosomal hydrolases. Such a systemic increase in lysosomal enzymes could result from much higher local concentrations of enzymes in the affected tissues.;To identify potential mechanisms responsible for the increase in lysosomal enzymes the subcellular localization of Ocrl was investigated by fluorescence microscopy. Ocrl was localized to endosomas and Golgi membranes along with clathrin, the mannose 6-phosphate receptor, and the early endosomal antigen 1. Binding assays show that Ocrl interacts with the clathrin terminal domain and the clathrin adaptor protein AP-2. These findings suggest a role for Ocrl in endosomal receptor trafficking and sorting, including sorting of the mannose 6-phosphate receptor, which is responsible for transporting lysosomal enzymes.;The tissues affected in Lowe syndrome depend on proper function of highly polarized cells raising the possibility that Ocrl is involved in polarized membrane trafficking. Mutations in the basolaterally localized NBC1 transporter lead to a phenocopy of Lowe syndrome, giving a strong genefc link between Ocrl and NBC1. The polarized distribution of NBC1 was analyzed in polarized MDCK cells depleted of Ocrl in an attempt determine the pathophysiology of Lowe syndrome.;A second focus of this thesis involved the cloning and initial characterization of the type I and type II phosphatidylinositol-4,5-bisphosphate 4-phosphatases, the first two mammalian enzymes found to hydrolyze the 4-phosphate of PtdIns-4,5-P 2. Both enzymes are ubiquitously expressend, associate with lysosomal membranes in cells, and appear to regulate the degradation of EGF receptors.